Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor

International Journal of Molecular Sciences

Article Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor

Marissa L. Maciej-Hulme 1,*,† , Eamon Dubaissi 2, Chun Shao 3, Joseph Zaia 3 , Enrique Amaya 2 , Sabine L. Flitsch 4 and Catherine L. R. Merry 1,*,‡

1 Materials Science Centre, School of Materials, The University of Manchester, Grosvenor St., Manchester M1 7HS, UK 2 Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Michael Smith Building, The University of Manchester, Oxford Road, Manchester M13 9PT, UK; [email protected] (E.D.); [email protected] (E.A.) 3 Center for Biomedical Mass Spectrometry, Boston University School of Medicine, 670 Albany Street, Boston, MA 02118, USA; [email protected] (C.S.); [email protected] (J.Z.) 4 School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK; [email protected] * Correspondence: [email protected] (M.L.M.-H.); [email protected] (C.L.R.M.) † Current address: Department of Nephrology, Radboudumc, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands. ‡ Current address: Nottingham Biodiscovery Institute, School of Medicine, University of Nottingham, Citation: Maciej-Hulme, M.L.; Nottingham NG7 2RD, UK. Dubaissi, E.; Shao, C.; Zaia, J.; Amaya, E.; Flitsch, S.L.; Merry, C.L.R. Abstract: The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental pro- Selective Inhibition of Heparan cesses. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors Sulphate and Not Chondroitin can be used to perturb HS function and these compounds provide cheap alternatives to genetic Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor. Int. J. manipulation methods. However, existing chemical inhibition methods for HS also interfere with Mol. Sci. 2021, 22, 6988. https:// chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method doi.org/10.3390/ijms22136988 for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Academic Editors: Chiara Schiraldi, Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Donatella Cimini and Annalisa Conversely, no adverse effect on CS production was observed. The inhibition was transient and La Gatta dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this Received: 24 May 2021 small molecule can be used as a tool for HS inhibition in biological systems. Accepted: 19 June 2021 Published: 29 June 2021 Keywords: heparan sulfate; azido sugar; glycosaminoglycan; carbohydrate biosynthesis; small molecule inhibitor; biorthogonal chemistry Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Heparan sulphate (HS) is a prevalent glycosaminoglycan (GAG) attached to protein cores (proteoglycans) on the cell surface of almost every cell type. HS proteoglycans form an integral part of the extracellular matrix with important roles in development [1], home- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ostasis [2,3] and disease [4,5]. HS is involved in cell-cell and cell-matrix communication, This article is an open access article fine-tuning cellular responses to the extracellular milieu. distributed under the terms and HS biosynthesis consists of a repeating disaccharide unit structure of glucuronic conditions of the Creative Commons acid–N-acetylglucosamine (GlcA-GlcNAc) polymerised by the exostoses enzyme complex Attribution (CC BY) license (https:// (EXT1/2) from UDP-GlcA and UDP-GlcNAc active nucleotide donor sugars [6–8]. During creativecommons.org/licenses/by/ this process the N-deacetylase/N-sulphotransferase (NDST) enzymes work in tandem to 4.0/). begin modification of the nascent chain. The NDST enzymes can replace the acetyl group

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Int. J. Mol. Sci. 2021this, 22 process, 6988 the N-deacetylase/N-sulphotransferase (NDST) enzymes work in tandem to 2 of 11 begin modification of the nascent chain. The NDST enzymes can replace the acetyl group on GlcNAc with a sulphate [9], often providing the gateway step for further modifications of the chain. Additionally, the NDSTs are also involved in control of HS chain length [10] with NDST shownon GlcNActo be co- withlocalised a sulphate with EXT2 [9], often [11]. providing During extension the gateway of the step backbone, for further modifications several other chemicaof thel chain.modifications Additionally, are possible, the NDSTs resulting are also in fine involved patterning in control of the of chain HS chain length [10] with NDST shown to be co-localised with EXT2 [11]. During extension of the backbone, where the functionality of HS is encoded. O-sulphotransferases (OSTs) modify the HS several other chemical modifications are possible, resulting in fine patterning of the chain chain at the 2-, 6- and 3-O position or epimerisation of GlcA to iduronic acid (IdoA) by where the functionality of HS is encoded. O-sulphotransferases (OSTs) modify the HS C5-epimerase can occur. Together, these enzymes contribute to HS functionality by influ- chain at the 2-, 6- and 3-O position or epimerisation of GlcA to iduronic acid (IdoA) by C5- encing the fine patterning of the chain [12]. epimerase can occur. Together, these enzymes contribute to HS functionality by influencing Despite its widespread role in biology, few chemical tools exist for the manipulation the fine patterning of the chain [12]. of HS function, with those available often interfering with chondroitin sulphate/dermatan Despite its widespread role in biology, few chemical tools exist for the manipulation sulphate (CS/DS) pathways simultaneously. Methods to ablate HS exist via targeted ge- of HS function, with those available often interfering with chondroitin sulphate/dermatan netic deletion of biosynthetic HS enzymes [6,7]. However, genetic manipulation is costly sulphate (CS/DS) pathways simultaneously. Methods to ablate HS exist via targeted and labour intensive with embryonic lethality in null mutant animals [7], posing chal- genetic deletion of biosynthetic HS enzymes [6,7]. However, genetic manipulation is lenges for post-embryoniccostly and labouranalysis. intensive In contrast, with chemical embryonic approaches lethality inoffer null cheap, mutant user animals- [7], posing friendly alternatives,challenges which for either post-embryonic perturb sulphation analysis. of Inthe contrast, chain [13,14] chemical or compete approaches with offer cheap, user- endogenous substratesfriendly involved alternatives, in HS which assembly, either such perturb as amino sulphation sugar of derivatives the chain [[15,16]13,14]or compete with and mimics of tetrasaccharideendogenous substrates linkages [17 involved–20]. However, in HS assembly, the additional such as effect amino on sugar CS/DS derivatives [15,16] synthesis can complicateand mimics data of interpretation tetrasaccharide particularly linkages [17 when–20]. both However, GAGs the are additional displayed effect on CS/DS on the proteoglycansynthesis of inte canrest complicate [21]. data interpretation particularly when both GAGs are displayed Azido sugarson theand proteoglycan other bio-orthogonal of interest chemistry [21]. approaches have been demonstrated as useful functionalisedAzido sugars chemical and other probes bio-orthogonal to label N-glycans chemistry [22] approaches, O-GlcNAc have modi- been demonstrated fications [23], mucinas useful type O functionalised-GalNAc glycans chemical [24] and probes sialic toacid label moietiesN-glycans [25]. Tetra [22],- O-GlcNAcacet- modifica- ylated N-azidoacetylgalactosaminetions [23], mucin type (Ac O-GalNAc4GalNAz) glycanscan be metabolically [24] and sialic converted acid moieties to UDP [25].- Tetra-acetylated GlcNAz and UDPN-azidoacetylgalactosamine-GalNAz via the GalNAc salvage (Ac4GalNAz) pathway can [23] be metabolically, potentiating converted its use in to UDP-GlcNAz GAG synthesis (andFigure UDP-GalNAz 1). via the GalNAc salvage pathway [23], potentiating its use in GAG synthesis (Figure1).

Figure 1. Figure(A) Structure 1. (A) Structure of tetra-acetylated of tetra-acetylatedN-azidoacetylgalactosamine. N-azidoacetylgalactosamine. (B) Schematic (B) Schematic of biological of biological azido sugar precursor productionazido for GAGsugar synthesis.precursor production Ac4GalNAz for travels GAG synthesis. across the Ac cell4GalNAz membrane travels and across enters the the cell cytoplasm. membrane Endogenous deacetylasesand removeenters the the cytoplasm acetyl protective. Endogenous groups deacetylases leaving GalNAz, remove ready the toacetyl enter protec the GalNActive groups salvage leaving pathway. After a cascadeGalNAz, of enzymes, ready both to enter UDP-GalNAz the GalNAc and salvage UDP-GlcNAz pathway. After are produced, a cascade whichof enzymes, target both CS/DS UDP and- potentially HS biosynthesisGalNAz respectively. and UDP-GlcNAz are produced, which target CS/DS and potentially HS biosynthesis respectively. The azido sugar nucleotide donors mimic UDP-GalNAc and UDP-GlcNAc, required for CS/DS and HS biosynthesis respectively. Recently, the EXT1/2 enzyme complex has been shown to utilise UDP-GlcNAz as a substrate for the addition of GlcNAz to the

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Int. J. Mol. Sci. 2021, 22, 6988 The azido sugar nucleotide donors mimic UDP-GalNAc and UDP-GlcNAc, required3 of 11 for CS/DS and HS biosynthesis respectively. Recently, the EXT1/2 enzyme complex has been shown to utilise UDP-GlcNAz as a substrate for the addition of GlcNAz to the non- reducingnon-reducing termini termini of heparan of heparan sulphate sulphate chains chains in vitroin vitro[26]. [However26]. However in vivoin, vivothere, there is the is potentialthe potential that that UDP UDP-GlcNAz-GlcNAz could could interfere interfere with with the the interaction interaction or or activity activity of of the the HS HS polymerisationpolymerisation machinery machinery ( (EXT/NDSTEXT/NDST enzymes enzymes)) due due to to the the location location of of the the azido azido group group situatedsituated on on the the acetyl acetyl position position of of the the GlcNAc GlcNAc residue, residue, thereby thereby producing producing an an inhibitory inhibitory effecteffect on on the the biosynthetic biosynthetic pathway. Therefore,Therefore, wewe soughtsought to to extend extend and and validate validate the the use use of of Ac4GalNAz treatment as a potential novel, small chemical inhibitor of HS synthesis. Ac4GalNAz treatment as a potential novel, small chemical inhibitor of HS synthesis.

22.. Results Results and and Discussion Discussion ChineseChinese hamster hamster ovary ovary (CHO (CHO)) cells cells were were treated treated with withdifferent different concentrations concentrations (7–35 µM(7–35) ofµ AcM)4GalNAz. of Ac4GalNAz. No lethal No effect lethal on effect the cells on thewas cells observed. was observed. Cell surface Cell HS surface was ana- HS lysedwasanalysed using flow using cytometry. ﬂow cytometry. A reduction A reduction in anti-(10E4 in anti-(10E4)) HS antibody HS staining antibody was staining observed was atobserved the cell surface at the cell in response surface in to response increasing to increasingAc4GalNAz Ac concentration4GalNAz concentration (Figure 2). (Figure 2).

FigureFigure 2. 2. FlowFlow cytometric cytometric analysis of Ac44GalNAzGalNAz-treated-treated CHOs.CHOs. CellsCells werewere treatedtreated with with 7–35 7–35µ µMM Ac Ac4GalNAz4GalNAz for for 24–48 24–48 h, h, or for the first 24 h, then 24 h without Ac4GalNAz (24 h rescue) and analysed for cell surface anti-HS (10E4) reactivity. or for the first 24 h, then 24 h without Ac4GalNAz (24 h rescue) and analysed for cell surface anti-HS (10E4) reactivity. Purple infilled, antibody control. Green trace, Ac4GalNAz-treated cells. Inset, experimental controls: purple infilled, antibody control; green trace, vehicle-treated; pink trace, untreated.

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Int. J. Mol. Sci. 2021, 22, 6988 4 of 11 Purple infilled, antibody control. Green trace, Ac4GalNAz-treated cells. Inset, experimental controls: purple infilled, antibody control; green trace, vehicle-treated; pink trace, untreated.

To sustain the reduction of HS for longer periods, higher concentrations (35 µM) of To sustain the reduction of HS for longer periods, higher concentrations (35 µM) of azido sugar were required. Both 7 µM and 17.5 µM gave partial population decreases in azido sugar were required. Both 7 µM and 17.5 µM gave partial population decreases in 10E4 staining and removal of Ac4GalNAz returned HS expression levels to match un- 10E4 staining and removal of Ac4GalNAz returned HS expression levels to match untreated treatedcell populations cell populations (24 h rescue), (24 h rescue indicating), indicating that the that effect the of effect the azidoof the sugar azido treatment sugar treat- on ment on HS was transient and reversible. Significantly less HS was present in 35 µM HS was transient and reversible. Signiﬁcantly less HS was present in 35 µM Ac4GalNAz Acconditions4GalNAz comparedconditions withcompared vehicle with control vehicle conditions control conditions with HS depletionwith HS depl displayingetion dis- a playingsigniﬁcant a significant dose-dependent dose-dependent decrease (Figuredecrease3A). (Figure 3A).

Figure 3. (A). Total relative abundance of HS from cell extracted samples. (B) Percentage chemical modiﬁcation contribution Figureof HS and3. (A (C).) Total percentage relative contribution abundance ofof HSHS disaccharidefrom cell extracted species samples. after RP-HPLC (B) Percentage separation chemical of 2-AMAC-tagged modification contribu- HS. Error tion of HS and (C) percentage contribution of HS disaccharide species after RP-HPLC separation of 2-AMAC-tagged HS. bars represent SEM of N = 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, student’s t test (two tailed). HexA, hexuronic acid (iduronic or glucuronic acid); 2S, 2-O-sulphate; 6S, 6-O-sulphate; NS, N-sulphate.

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Int.Error J. Mol. bars Sci. 2021 represent, 22, 6988 SEM of N = 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, student’s t test (two tailed). HexA, 5 of 11 hexuronic acid (iduronic or glucuronic acid); 2S, 2-O-sulphate; 6S, 6-O-sulphate; NS, N-sulphate.

Furthermore, HS biosynthesis was perturbed as a subtle, but significant change in Furthermore, HS biosynthesis was perturbed as a subtle, but signiﬁcant change in disaccharide composition (Figure 3B,C) showing alterations in the sulphation of the chain disaccharide composition (Figure3B,C) showing alterations in the sulphation of the chain (increase in 6-O-sulphation and decrease 2-O-sulphation), reminiscent of GAG biosyn- (increase in 6-O-sulphation and decrease 2-O-sulphation), reminiscent of GAG biosynthetic thetic enzyme mutants [27,28]. To elucidate changes in HS chain length, CHO cell cultures enzyme mutants [27,28]. To elucidate changes in HS chain length, CHO cell cultures were radiolabelled with 3H-glucosamine alongside treatment with Ac4GalNAz and HS were radiolabelled with 3H-glucosamine alongside treatment with Ac GalNAz and HS populations from cell extracts were purified as previously described [29]. 4Total GAG syn- populations from cell extracts were puriﬁed as previously described [29]. Total GAG thesis was normalised to protein level. Radiolabelled studies showed a dose dependent synthesis was normalised to protein level. Radiolabelled studies showed a dose dependent decrease in the quantity (Figure 4) and chain length of HS (Table 1) in Ac4GalNAz-treated decrease in the quantity (Figure4) and chain length of HS (Table1) in Ac 4GalNAz-treated cells compared to control. cells compared to control.

Figure 4. Total GAG synthesis normalised to protein levels from CHO-K1 cells. Figure 4. Total GAG synthesis normalised to protein levels from CHO-K1 cells.

TableTable 1. 1.ChainChain length length of ofradiolabelled radiolabelled HS HS and and CS/DS. CS/DS.

SecretedSecreted Modal Modal Size Size ( (kDa)kDa) Cell Cell Extract Extract Modal Modal Size Size ( (kDa)kDa) ConditionCondition HSHS CS/DS HS HS CS/DS CS/DS VehicleVehicle control control 2222 32 8.5 8.5 38 38 7 µM7 µM Ac Ac4GalNAz4GalNAz 1212 22 6.9 6.9 31 31 35 µM Ac GalNAz 7 32 7.5 32 35 µM Ac4GalNAz4 7 32 7.5 32

TheThe marked marked decrease decrease in inchain chain length length observed observed in inboth both secreted secreted HS HS and and cell cell-derived-derived HSHS populations populations after after Ac Ac4GalNAz4GalNAz treatment treatment (Table (Table 1)1 )suggests suggests that that early early termination termination of of chainchain synthesis synthesis was was responsible responsible for for the the depletion depletion of ofHS HS at atthe the cell cell surface surface observed observed in inflow flow cytometriccytometric experiments experiments (Figure (Figure 2)2.). DespiteDespite significant significant changes changes to toHS HS chain chain synth synthesis,esis, no no incorporation incorporation of ofazido azido groups groups waswas detected detected in inHS HS chains chains (data (data not notshown shown),), suggesting suggesting that thateither either GlcNAz GlcNAz was not was in- not corporatedincorporated into intothe thechain chain or that or that the theazide azide was was potentially potentially removed removed by by NDST NDST activity activity duringduring HS HS synthesis. synthesis. DueDue to toconvergence convergence in their in their synthetic synthetic pathways pathways (Figure (Figure 1) and1) the and utilisation the utilisation of com- of moncommon precursors, precursors, chemical chemical inhibitors inhibitors usually usually affect both affect HS both and HS CS/DS and CS/DSGAGs indiscrimi- GAGs indis- nately,criminately, therefore therefore we assessed we assessed whether whether CS/DS CS/DS synthesis synthesis was wasalso also inhibited inhibited by bythe the same same metabolicmetabolic labelling labelling strategy. strategy. Azido Azido sugar sugar labelling labelling of ofCS CS proteoglycans proteoglycans using using GalNAz GalNAz has has beenbeen described described previously previously [30,31] [30,31, ],but but examination examination of of the the biosynthesis biosynthesis of of CS/DS CS/DS was was not not reported. No changes in CS/DS composition (Figure5), chain length (Table1) or quantity were observed (Figure4), suggesting that the inhibitory effect of Ac 4GalNAz treatment was specific to HS synthesis.

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reported. No changes in CS/DS composition (Figure 5), chain length (Table 1) or quantity were observed (Figure 4), suggesting that the inhibitory effect of Ac4GalNAz treatment Int. J. Mol. Sci. 2021, 22, 6988 was specific to HS synthesis. 6 of 11

Figure 5. CS/DS disaccharide analysis from (A) CHO-K1 cell extracts and (B) cell-conditioned medium. Percentage contributionFigure 5. CS/DS of CS/DS disaccharide disaccharide analysis species from ( afterA) CHO SAX-HPLC-K1 cell extracts of radiolabelled and (B) cell preparations.-conditioned Other medium CS/DS. Percentage disaccharide con- speciestribution were of CS/DS not detected. disaccharide HexA, species hexuronic after acid SAX (iduronic-HPLC of or radiolabelled glucuronic acid). preparations. 4S, 4-O-sulphate; Other CS/DS 2S, 2-O-sulphate. disaccharide species were not detected. HexA, hexuronic acid (iduronic or glucuronic acid). 4S, 4-O-sulphate; 2S, 2-O-sulphate. Ac4ManNAz has also been reported to label CS proteoglycans, however the presence of CS-specificAc4ManNAz labelling has also on thebeen proteoglycan reported to lab wasel notCS proteoglycans, investigated [32 however]. Notably, the no presence NDST enzymesof CS-specific are associated labelling on with the CS/DS proteoglycan synthesis was and not theinvestigated acetyl group [32] of. Notably, GalNAc no remains NDST unmodified,enzymes are whereasassociated HS with biosynthesis CS/DS synthesis specifically and involvesthe acetyl removal group of theGalNAc acetyl remains and N- sulphationunmodified of, whereas GlcNAc. HS This biosynthesis process, at leastspecifically in part, involves controls HSremoval chain of length the acetyl [11] possibly and N- viasulphation NDST interaction of GlcNAc. with This EXT process, co-polymerase, at least in althoughpart, controls the mechanism HS chain length is still unclear.[11] possibly EXT enzymevia NDST activity interactionin vitro withhas EXT been co demonstrated-polymerase, to although utilise UDP-GlcNAz the mechanism as ais substrate still unclear. [26], suggestingEXT enzyme that activity extension in vitro of has the been HS chain demonstrated by the EXTs to utilise is likely UDP to- remainGlcNAz unaffected as a substrate by GlcNAz[26], suggestingin vivo. Thus,that extension we hypothesise of the HS that chain the presence by the EX ofTs GlcNAz is likely interferes to remain with unaffected normal NDSTby GlcNAz function, in vivo thereby. Thus, we inhibiting hypothesise HS synthesis that the presence and resulting of GlcNAz in truncated interferes HS with chains. nor- Importantly,mal NDST function, upregulation thereby of inhibiting NDST1 has HS been synthesis associated and resulting with chemoresistance in truncated HS in chains. breast cancerImportantly, [33] and upregulation upregulated of NDST1 NDST1 activity has been increases associated HS biosynthesis with chemoresistance [11]. Thus, selectivein breast strategiescancer [33] for and inhibition upregulated of HS NDST1 activity, activity such increases as this one, HS maybiosynthesis have therapeutic [11]. Thus, potential selective alongsidestrategies currentfor inhibition treatment of HS options activity, where such HS as is athis known one, driver may have of chemoresistance therapeutic potential and/or tumouralongside growth. current treatment options where HS is a known driver of chemoresistance and/orFinally, tumour since growth. HS has been also demonstrated to play important roles in develop- mentFinally, [1], we soughtsince HS to has test been the ability also demonstrated of the azido sugar to play to important inhibit HS roles in a model in development organism. Therefore,[1], we sought a well-characterised to test the ability and of widely the azido used sugar developmental to inhibit HS biology in a vertebratemodel organism. model (Therefore,Xenopus tropicalis a well-characterised) was utilised and [34 ].widely Following used developmental treatment with biology the inhibitor, vertebrate the model abundance(Xenopus of totaltropicalis HS in) was Ac4 GalNAz-treatedutilised [34]. Following embryos treatment was decreased with comparedthe inhibitor, with the controls abun- (Figure6A), confirming that this small, soluble inhibitor can be used in both organismal dance of total HS in Ac4GalNAz-treated embryos was decreased compared with controls and cell culture-based experiments to inhibit HS synthesis. The Ac4GalNAz-treated Xenopus embryos displayed a phenotypic short stature (Figure6B) accompanied with irregular somite boundaries and abnormal skeletal muscle orientation in a dose dependent manner, resulting in gross disorganization of the tail structure and tail kinks (Supplementary Figure S1). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 11

(Figure 6A), confirming that this small, soluble inhibitor can be used in both organismal and cell culture-based experiments to inhibit HS synthesis. The Ac4GalNAz-treated Xenopus embryos displayed a phenotypic short stature (Fig- ure 6B) accompanied with irregular somite boundaries and abnormal skeletal muscle ori- Int. J. Mol. Sci. 2021, 22, 6988 7 of 11 entation in a dose dependent manner, resulting in gross disorganization of the tail structure and tail kinks (Supplementary Figure S1).

Figure 6. Ac4GalNAz treatment of Xenopus embryos. (A) Total HS (pmol) per embryo. (B) Embryo stature measure- Figure 6. Ac4GalNAz treatment of Xenopus embryos. (A) Total HS (pmol) per embryo. (B) Embryo stature measurements ments of the tail extension (µm). + Ac4GalNAz, 500 pmol injection and 500 µM soaking; + Ac4GalNAc, 500 pmol of the tail extension (µm). + Ac4GalNAz, 500 pmol injection and 500 µM soaking; + Ac4GalNAc, 500 pmol injection and injection and 500 µM soaking. * p ≤ 0.05, *** p ≤ 0.001 + Ac GalNAz vs. Wildtype. Oneway ANOVA with Turkey 500 µM soaking. * p ≤ 0.05, *** p ≤ 0.001 + Ac4GalNAz vs. Wildtype.4 Oneway ANOVA with Turkey post-hoc comparison. post-hoc comparison. Interestingly, similar developmental abnormalities have been observed in UDP-4-az- Interestingly, similar developmental abnormalities have been observed in UDP-4- ido-4-deoxyxylose-treated zebrafish [17], where GAG synthesis (CS and HS) was broadly azido-4-deoxyxylose-treated zebraﬁsh [17], where GAG synthesis (CS and HS) was broadly targeted, preventing elongation of either type of GAG. Targeted application of targeted, preventing elongation of either type of GAG. Targeted application of Ac4GalNAz Ac4GalNAz as a small, selective HS inhibitor thus provides independent evaluation of the as a small, selective HS inhibitor thus provides independent evaluation of the role of HS in role of HS in biological systems where transient knock down of HS biosynthesis is desired. biological systems where transient knock down of HS biosynthesis is desired.

3. Conclusions3. Conclusions We propose that a common sugar analogue, Ac4GalNAz, can be applied as a small, We propose that a common sugar analogue, Ac4GalNAz, can be applied as a small, solublesoluble and and reversible reversible chemical chemical inhibitor inhibitor of HS, of HS, which which does does not not affect affect CS/DS CS/DS biosynthesis, biosynthesis, offering a new tool for HS inhibition. Ac4GalNAz can be synthesised from inexpensive offering a new tool for HS inhibition. Ac4GalNAz can be synthesised from inexpensive compoundscompounds [35 ][35] and and is commercially is commercially available. available. Using Using this this strategy, strategy, HS HS inhibition inhibition can can be be achievedachieved in cell-basedin cell-based assays assays and and in whole in whole organisms. organisms. The The effect effect of Ac of4 GalNAzAc4GalNAz on HSon HS productionproduction is transient is transient (Figure (Figure2), enabling 2), enabling ﬂexible flexible application application and removal and removal in experiments in experi- withoutments the without need for the gene need manipulation. for gene manipulation. This novel This selective novel HS selective inhibitor HS therefore inhibitor may therefore be usedmay to probe be used HS biologyto probe in HS separation biology from in separation CS/DS to from identify CS/DS HS-mediated to identify mechanisms HS-mediated in biologicalmechanisms systems in biological for further systems investigation. for further investigation.

4. Materials4. Materials and and Methods Methods 3 4.1.4.1. Cell Cell Culture, Culture, Ac4 AcGalNAz4GalNAz and and D-[6- D-[6H]-Glucosamine-3H]-Glucosamine Treatment Treatment ChineseChinese Hamster Hamster Ovary-K1 Ovary- (CHO-K1)K1 (CHO-K1) cells cells (gifted (gifted from from the the Esko Esko lab) lab) were were cultured cultured ◦ at 37at 37C/5% °C/5% CO CO2 humidiﬁed2 humidified conditions conditions inin Dulbecco’sDulbecco’s ModiﬁedModified Eagle Medium: F12 F12 Nu- Nutrienttrient Mix (Ham) (Ham) media media (Invitrogen (Invitrogen,, Loughborough, Loughborough, UK UK)) supplemented supplemented with with 10% 10% v/v fe- v/vtalfetal bovine bovine serum serum (FBS) (FBS) (batch (batch-tested,-tested, Biosera) Biosera) and and 2 mM 2 mM L-glutamine L-glutamine (PAA). (PAA). Cell Cellculture culturemedium medium was wassupplemen supplementedted with with sugars sugars dissolved dissolved in dimethylsulfoxide in dimethylsulfoxide (DMSO): (DMSO): Ac4AcGalNAz4GalNAz (Molecular (Molecular Probes, Probes Loughborough,, Loughborough, UK), UK Ac),4 GalNAcAc4GalNAc (gifted (gifted from from the the Flitsch Flitsch group). For radioactive experiments, CHO medium was supplemented with 50 µCi D- [6-3H]-glucosamine hydrochloride (Perkin Elmer, Llantrisant, UK). CHO-K1 cells were seeded at 40,000 cells/cm2 and then cultured for 48 h for metabolic incorporation of the radiolabelled sugar. Int. J. Mol. Sci. 2021, 22, 6988 8 of 11

4.2. Flow Cytometry To preserve the cell surface, non-enzymatic cell dissociation buffer (Gibco, Lough- borough, UK) was used to remove CHOs from tissue culture plastic. After washing with phosphate buffered saline (PBS), cells were incubated with anti-HS (F58–10E4) (1:200, Amsbio, Abingdon, UK) in 0.2% (w/v) bovine serum albumin (BSA), followed by Alex- aFluor goat anti-mouse IgM (κ)-488 (1:1000, Molecular Probes, Loughborough, UK). Cells were ﬁxed with 1% PFA for 10 min at room temperature before analysis on a Cyan ADP cytometer (Beckman Coulter, High Wycombe, UK) using CellQuest Pro software).

4.3. GAG Collection and Puriﬁcation Cell membranes were dispersed with 1% Triton X-100 in PBS with gentle agitation for 1–2 h. Proteins were digested with 100 µg/mL Pronase (Streptomyces griseus, Roche, Welwyn Garden City, UK) for 4 h at 37 ◦C. Diethylaminoethyl (DEAE) anion-exchange chromatography for GAG preparations with step elution of HS and CS/DS using 1.5 M NaCl was used to isolate GAG material as previously described [36], with the exception of the radiolabelled GAG preparations where gradient anion exchange chromatography (0–1.5 M NaCl) was used. GAG samples were desalted using PD10 columns (GE Healthcare, Amersham, UK) and lyophilised.

4.4. HS and CS Chain Length Analysis Purified D-[6-3H]-radiolabelled HS or CS material was treated with 50 mM NaOH/1 ◦ M NaBH4 at 45 C for 48 h to cleave the protein stub from the xylose residue. Samples were neutralized with glacial acetic acid and then separated on Sepharose CL-6B columns in 0.2 M ammonium bicarbonate at a flow rate of 0.2 mL/min. 1 mL fractions were collected in pony vials (Sigma, Gillingham, UK) and 2 mL Optimax scintillation fluid (Perkin Elmer, Llantrisant, UK) was added. Samples were sealed and shaken before processing for 3H radioactivity (counts per minutes) using a liquid scintillation counter (Wallac 1409, Beckman Coulter, High Wycombe, UK). Modal chain length was estimated by comparison of Kav values with a calibration curve [37].

4.5. Generation of GAG Disaccharide Species Puriﬁed GAG samples were digested either with 2 mIU of each heparinase I–III (Iduron) in 0.1 M sodium acetate, 0.1 mM calcium acetate, pH 7.0 or with chondroitinase ABC (Amsbio, Abingdon, UK) in 50 mM tris, 50 mM NaCl, pH 7.9 for 16 h at 37 ◦C. For radiolabelled preparations, the disaccharides were separated from oligosaccharides via Superdex-30 chromatography.

4.6. 2-AMAC Labelling and RP-HPLC Separation of HS Disaccharides HS disaccharides were labelled with 2-aminoacridone (2-AMAC) and separated using RP-HPLC as previously described using correction factors for the batch of 2-AMAC utilised [38,39]. Data was also used to calculate the HS sulphation modiﬁcation.

4.7. Strong Anion Exchange (SAX)-HPLC Separation of CS Disaccharides Samples of 5000 cpm digested D-[6-3H]-CS disaccharides were separated on a Hypersil 5 µm SAX column (Thermo Scientiﬁc, Loughborough, UK) with a gradient of 0.15 M–0.7 M NaCl pH 3.5 over 47 min at a ﬂow rate of 1 mL/min.

4.8. Sugar Microinjection and Incubation of Xenopus Embryos Fertilised NF stage 1 embryos in injection buffer (1% (w/v) Ficoll in 0.1× Modiﬁed Marc Ringers, (MMR), pH 8.0) were injected with 1–5 nL of 500–1000 picomoles Ac4GalNAz or Ac4GalNAc (dissolved in 0.2 mM KCl) into the cytoplasm using a heat-pulled borosilicate glass capillary injection needle (1 mm × 0.78 mm, Harvard apparatus, Holliston, MA, USA). Embryos were left to recover in injection buffer for 1–2 h (stage 7–8) at 28 ◦C before they were transferred to fresh agarose-coated dishes containing a bath of 0–500 µM Ac4GalNAz Int. J. Mol. Sci. 2021, 22, 6988 9 of 11

◦ or Ac4GalNAc in 0.01 × MMR solution. Embryos were incubated at 23 C (prior to gastrulation) for the ﬁrst day of development, then at 25 ◦C and transferred to fresh sugar/0.01 × MMR conditions daily.

4.9. Puriﬁcation of Xenopus HS Embryos were lyophilised and ground in a pestle and mortar with 1 mL of PBS before addition of 1 mg/mL Pronase in 50 mM Tris/HCl pH 8.0, 1 mM CaCl2, 1% Triton X-100. Proteins were digested for 16 h at 55 ◦C, then a further 0.5 mg Pronase was added and the digestion continued overnight. Pronase was heat-inactivated at 100 ◦C for 10 min and samples were then treated with 2 µL of 2 M MgCl2 and 0.5 µL Benzonase Nuclease (300 mU, Sigma, Gillingham, UK) at 37 ◦C for 3 h before adjustment to 0.5 M NaOH and mixing overnight. Formic acid was used to adjust the pH to 5.0 prior to centrifugation at 13,000 rpm. The supernatant was diluted with HPLC grade water and applied to DEAE anion exchange chromatography as described in [36] with the following alterations: DEAE beads were washed only with HPLC grade water prior to sample application and samples were eluted with 1 M NaCl, 20 mM NaOAc pH 6.0. The eluent was desalted using PD10 columns according to the manufacturer’s instructions.

4.10. Mass Spectrometry Analysis of Xenopus HS Disaccharides Xenopus HS disaccharides were diluted in 200 µL HPLC grade water and centrifuged at 12,000 rpm for 10 min to remove insoluble material. Residual salts and/or proteins were removed from the supernatant using size-exclusion chromatography (Beckman SEC ofﬂine fractionate), followed by further clean up using a porous graphite carbon C-18 TopTip (Glygen) prior to Liquid Chormatography-Mass Spectrometry using a Dionex GlycanPac AXH-1 (1 mm × 15 cm) (ThermoFisher, Walton, MA, USA) on an Agilent QTOF 6520 in negative mode, with an acquisition range of 100–1700 m/z. Heparin disaccharide I-P sodium salt (∆UA2S-GlcNCOEt6S) (V-labs, Dextra Laboratories, Reading, UK) was spiked into all samples as an external standard to monitor the spray conditions and used for normalization between samples.

4.11. Whole Mount Antibody Fluorescent Imaging Embryos were ﬁxed in 0.1 M 3-(N-Morpholino)propane sulfonic acid (MOPS) pH 7.4, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA), ◦ 1 mM MgSO4, 3.7% (v/v) formaldehyde for 16 h at 4 C before dehydration with 100% methanol. Embryos were then rehydrated by gradient dilution of the methanol with H2O and antibody staining was performed as previously described [40] using mouse 12/101 IgG1 (1:200, Developmental Studies Hybridoma Bank) followed by AlexaFluor goat anti-mouse IgG (H + L)-594 (1:500, Molecular Probes). Embryos were imaged using a glass-bottomed dish (MatTek Corporation, Bratislava, Slovakia) and imaged by confocal microscopy using an Olympus Fluoview FV1000 (Olympus, Southend-on-Sea, UK).

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22136988/s1, Figure S1: Wholemount ﬂuorescence of skeletal muscle in stage 39 NF Xenopus tropicalis tadpoles after the injection of sugars. Author Contributions: M.L.M.-H., C.L.R.M.; Methodology: M.L.M.-H.; Validation: M.L.M.-H., C.S.; Formal analysis: M.L.M.-H.; Investigation: M.L.M.-H., C.S., E.D.; Writing—original draft: M.L.M.-H.; Writing—review & editing: M.L.M.-H., C.L.R.M., S.L.F., E.A., E.D., J.Z., C.S.; Supervision: J.Z., S.L.F., E.A., C.L.R.M.; Project administration: C.L.R.M.; Funding acquisition: E.A., C.L.R.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a BBSRC DTC Studentship Grant No. 978724 and MRC grant (MR/L007525/1). Institutional Review Board Statement: Experiments involving Xenopus embryos were approved by the local ethics committee and the Home Ofﬁce (PPL 70/7648). Int. J. Mol. Sci. 2021, 22, 6988 10 of 11

Informed Consent Statement: Not applicable. Acknowledgments: The authors would like to thank Robert Lea for technical support throughout the generation of the Xenopus data, Santanu Mandal for per-acetylation of GalNAc (Ac4GalNAc) and Jeffrey Esko for the CHO-K1 cells. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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